A Replication-Competent HIV Clone Carrying GFP-Env Reveals Rapid Env Recycling at the HIV-1 T Cell Virological Synapse

HIV-1 infection is enhanced by cell–cell adhesions between infected and uninfected T cells called virological synapses (VS). VS are initiated by the interactions of cell-surface HIV-1 envelope glycoprotein (Env) and CD4 on target cells and act as sites of viral assembly and viral transfer between cells. To study the process that recruits and retains HIV-1 Env at the VS, a replication-competent HIV-1 clone carrying an Env-sfGFP fusion protein was designed to enable live tracking of Env within infected cells. Combined use of surface pulse-labeling of Env and fluorescence recovery after photobleaching (FRAP) studies, enabled the visualization of the targeted accumulation and sustained recycling of Env between endocytic compartments (EC) and the VS. We observed dynamic exchange of Env at the VS, while the viral structural protein, Gag, was largely immobile at the VS. The disparate exchange rates of Gag and Env at the synapse support that the trafficking and/or retention of a majority of Env towards the VS is not maintained by entrapment by a Gag lattice or immobilization by binding to CD4 on the target cell. A FRAP study of an Env endocytosis mutant showed that recycling is not required for accumulation at the VS, but is required for the rapid exchange of Env at the VS. We conclude that the mechanism of Env accumulation at the VS and incorporation into nascent particles involves continuous internalization and targeted secretion rather than irreversible interactions with the budding virus, but that this recycling is largely dispensable for VS formation and viral transfer across the VS.

B2M gene knockout in HEK293T cells by non-viral delivery of CRISPR-Cas9 for the induction of universal cell

Allogeneic stem cells have been introduced as a potential approach to generate grafts in regenerative medicine. But the clinical usage of them is limited due to the risk of immune rejection that is induced by the incompatibility of human leukocyte antigens (HLAs) between donors and recipients. To overcome this limitation, we knocked out the β2 microglobulin (B2M) gene which is crucial for HLA class I expression, using CRISPR/Cas9 approach. Non-viral transfer of two gRNAs targeting exon 1 and intron 1 in B2M gene caused in large and predictable deletion in region between two loci, which was defined by sequencing and polymerase chain reaction (PCR). Furthermore, results revealed that roughly 11.11% and 22.22% of the GFP expressing cells reflecting a homozygous and heterozygous pattern of genomic modifications. We demonstrated that the dual guide RNA approach is a simple and efficient method for gene disruption. Significantly, these engineered hypoimmunogenic cells could be proposed as universal cells that are not distinguishable to the recipient immune system in cell therapy and transplantation.

Mesenchymal Stem Cells as a Gene Delivery Tool: Promise, Problems, and Prospects

The cell-based approach in gene therapy arises as a promising strategy to provide safe, targeted, and efficient gene delivery. Owing to their unique features, as homing and tumor-tropism, mesenchymal stem cells (MSCs) have recently been introduced as an encouraging vehicle in gene therapy. Nevertheless, non-viral transfer of nucleic acids into MSCs remains limited due to various factors related to the main stakeholders of the process (e.g., nucleic acids, carriers, or cells). In this review, we have summarized the main types of nucleic acids used to transfect MSCs, the pros and cons, and applications of each. Then, we have emphasized on the most efficient lipid-based carriers for nucleic acids to MSCs, their main features, and some of their applications. While a myriad of studies have demonstrated the therapeutic potential for engineered MSCs therapy in various illnesses, optimization for clinical use is an ongoing challenge. On the way of improvement, genetically modified MSCs have been combined with various novel techniques and tools (e.g., exosomes, spheroids, 3D-Bioprinting, etc.,) aiming for more efficient and safe applications in biomedicine.

Investigating the presence of SARS-CoV-2 on the Surfaces, Fomites, and in Indoor Air of a referral COVID-19 Hospital in a Middle Eastern Area

Coronavirus illness (COVID-19) is an immensely transmissible viral infectious disease caused by Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2). This study aimed to assess the presence of SARS-CoV-2 in the indoor air, on the surfaces, and the fomites, and in the indoor air of a COVID-19 referral hospital in Shiraz, Iran. Indoor air sampling was conducted utilizing a standard midget impinger contained 15 ml of viral transfer medium (VTM) equipped with a sampling pump with a flow rate of 10 L min− 1 for 60 minutes. Surfaces and fomites were sampled using sterile polyester swabs. The RNA of SARS-CoV-2 was detected in about 41.2% of indoor air and 32% of swab samples. Four out of the six (66.7%) indoor air samples up to a distance of 2 meters from the patient’s bed in intensive care units (ICU-1, ICU-3), accident and emergency (A&E-2), and negative pressure room were positive for SARS-CoV-2 RNA. All air samples within 2 to 5 meters from the patient’s bed were negative. This study’s results did not support the airborne SARS-CoV-2 transmission; However, it showed contamination of surfaces and fomites in the studied hospital’s wards.

SARS-CoV-2 in peritoneal swabs from asymptomatic patients undergoing emergency abdominal surgery

This is a case series of five patients with acute abdomen requiring surgery who tested positive for coronavirus disease 2019 (COVID-19) and were asymptomatic, with the purpose of detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in peritoneal fluid. Nasopharyngeal swab was done as a prerequisite for admission or prior to admission as part of random testing. Two methods of viral testing were employed: Xpert® Xpress SARS-CoV-2 (rapid test) and real-time reverse transcription polymerase chain reaction (RT-PCR). Either or both tests were done, with the former performed for patients requiring surgery immediately. Surgery was performed within 24–36 h from admission. Peritoneal fluid swabs were obtained for the detection of SARS-CoV-2 using RT-PCR test. Swabs were immediately placed in viral transfer media and delivered to the public health laboratory in an ice bag. SARS-CoV-2 was not detected in peritoneal swabs. Due to the limited number of patients, further studies are required; yet, protective measures should still be taken by surgeons when dealing with COVID-19 cases.

Presence of SARS-CoV-2 RNA on playground surfaces and water fountains

The possibility of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) transmission by fomites or environmental surfaces has been suggested. It is unclear if SARS-CoV-2 can be detected in outdoor public areas. The objective of the current study was to assess the presence of SARS-CoV-2 in environmental samples collected at public playgrounds and water fountains, in a country with high disease prevalence. Environmental samples were collected from six cities in central Israel. Samples were collected from drinking fountains and high-touch recreational equipment at playgrounds. Sterile pre-moistened swabs were used to collect the samples, put in viral transfer media and transferred to the laboratory. Viral detection was achieved by real-time reverse transcriptase–polymerase chain reaction, targeting four genes. Forty-three samples were collected from playground equipment and 25 samples from water fountains. Two of the 43 (4.6%) samples from playground equipment and one (4%) sample from a drinking fountain tested positive. It is unclear whether the recovery of viral RNA on outdoor surfaces also indicates the possibility of acquiring the virus. Adherence to environmental and personal hygiene in urban settings seems prudent.

Paired SARS CoV-2 Spike Protein Mutations Observed During Ongoing SARS-CoV-2 Viral Transfer from Humans to Minks and Back to Humans

A mutation analysis of a collection of SARS-CoV-2 genomes around the world via sequence, date, geographic location, and species has revealed a large number of variants from the initial reference sequence in Wuhan. It also reveals that humans infected with SARS-CoV-2 have infected mink populations in the Netherlands, Denmark, United States, and Canada. In these animals, a small set of mutations often in combination, in the spike protein receptor binding domain (RBD) has apparently transferred back into humans. The viral genomic mutations in minks observed in the Netherlands and Denmark show the potential for new mutations on the SARS-CoV-2 spike protein RBD to be introduced into humans by zoonotic transfer. Our data suggests that close attention to viral transfer from humans to farm animals and pets will be required to prevent build-up of a viral reservoir for future zoonotic transfer.

Recruitment of Env to the HIV-1 T cell virological synapse by targeted and sustained Env recycling

ABSTRACTHIV-1 infection is enhanced by cell-cell adhesions between infected and uninfected T cells called virological synapses (VS). VS are initiated by the interactions of cell-surface HIV-1 envelope glycoprotein (Env) and CD4 on target cells and act as sites of viral assembly and viral transfer between cells. To study the process that recruits and retains HIV-1 Env at the VS, a replication-competent HIV-1 clone carrying an Env-sfGFP fusion protein was designed to enable live tracking of Env within infected cells. Using surface pulse-labeling of Env and fluorescence recovery after photobleaching (FRAP) studies, we observed targeted accumulation and sustained recycling of Env between the endocytic recycling compartment (ERC) and the VS. We observed dynamic exchange of Env at the VS while the viral structural protein, Gag, was largely immobile at the VS. The disparate exchange rates of Gag and Env at the synapse indicate that retention of Env is not likely to be maintained by entrapment into an immobile Gag lattice or through immobilizing interactions with CD4 on the target cell. A FRAP study of an Env endocytosis mutant showed that recycling is required for the rapid exchange of Env at the VS. We conclude that the mechanism of Env accumulation at the VS and incorporation into nascent particles involves continuous internalization and targeted secretion rather than irreversible interactions with the budding virus.